Mentor: Andrea Kravats, Ph.D.

This study explores the effect of charged residues around the N-terminal domain on the peptide-binding activity of Grp94. The ER chaperone Grp94 is a key part of the cell's innate immune response, as it helps prevent protein aggregation and chaperones several viral proteins to T-cells. Previous studies have found that the majority of Grp94’s binding activity occurs at the N-terminal domain (Gidalevitz et al., Journal of Biological Chemistry, 2004). By studying mutant Grp94 proteins in which charged residues around the N-domain have been changed to neutral amino acid residues, we hope to learn more about the binding mechanism of the protein. The wild type [Grp94 WT] and mutant proteins [Grp94 H146A and Grp94 E141A-K142A] were expressed in E. coli cells and purified using affinity and size-exclusion chromatography. To determine the binding affinity of these proteins for the peptide VSV8, a fluorescent polarization assay was performed, and the Kd values were calculated for both the WT and mutant proteins. Results found that the Grp94 WT had a higher affinity for the peptide than both the Grp94 H146A and Grp94 E141A-K142A mutants. In addition, the Grp94 E141-K142A mutant showed a higher binding affinity than the H146A mutant. This indicates that overall, the removal of charged residues in the N-terminal domain decreased the binding activity of the protein. Furthermore, this shows that the removal of a charged amino acid at the 146 residue of the protein had a more significant impact on binding affinity than changes in the 141 and 142 residues.

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